Clear cell renal cell carcinoma, the most common subtype of kidney cancer, is characterized by mutations in genes encoding chromatin modifiers. One of these genes, SETD2, encodes the only methyltransferase capable of trimethylation of histone H3 lysine 36 (H3K36me3). In patients, mutation of SETD2 has been associated with decreased overall survival and time to recurrence. Previous work has shown that mutation of SETD2 in human tumors is associated with altered chromatin accessibility and wide-spread RNA processing defects. Common mutations in SETD2 include early frameshift/nonsense mutations, mutations in the catalytic SET domain and mutations in the SRI domain, which mediates the interaction between SETD2 and RNA polymerase. The central hypothesis of this proposal is that SETD2 acts as a tumor suppressor by regulating chromatin during transcription, and that disruption of this process promotes cancer through both transcriptional processing defects and widespread reorganization of chromatin packaging. Aim 1 will identify the effect of specific SETD2 mutations in the SET and SRI domains on cellular transformation and RNA processing. Cell lines have been engineered for SETD2 inactivation and expression of tumor-associated SETD2 mutants in the SET domain and SRI domain. These cells will be used to study proliferation, anchorage independent growth, and cell migration. These cells will also be examined for altered RNA processing, including aberrant splicing, altered exon utilization, and alternate transcription start/termination sites, using RNA-seq data and published and custom generated computational algorithms. Aim 2 will examine the effect of SETD2 mutation on H3K36me3 placement as well as overall chromatin organization. H3K36me3 localization will be examined by ChIP-seq in cell harboring SRI domain mutants. Cells will be tested for changes in chromatin organization by FAIRE-seq and MNAse-seq. RNA, ChIP, chromatin accessibility and nucleosome positioning data will then be integrated to explore the role of SETD2 and its effect on chromatin organization. Results from this study will further our understanding of the ability of SETD2 to suppress tumor development by maintaining epigenomic organization and transcriptomic fidelity.